1. Field of the Invention
The present invention relates to a rapid, sensitive assay for peroxidase. More particularly, the present invention relates to a composition, element and method for carrying out a simplified, fluorometric peroxidase assay useful for detecting low enzyme levels.
2. Description of the Prior Art
The oxidation of dichlorofluorescin by hydrogen peroxide to dichlorofluorescein, catalyzed by the enzyme peroxidase, has been used as an assay technique for hydrogen peroxide (Black, M. J. & Brandt, R. B., Anal, Biochem, 58:241 [1974]). A drawback of this technique is that since the dichlorofluorescin is unstable to air oxidation, it must be generated as needed by hydrolysis of diacetyldichlorofluorescin at alkaline pH followed by neutralization and dilution in the assay tube.
The assay of peroxidase, particularly at relatively low levels, is useful in certain immunoassay techniques wherein a peroxidase is used as the label for a ligand in a competitive binding or displacement immunological determination. Such assay techniques are described in U.S. Pat. No. 3,817,837 to Rubenstein et al; U.S. Pat. No. 3,154,090 to Schuurs et al and in Van Weeman, B. K. and Schuurs, A.H.W.M., BIOCHIMIE, 54:842 (1972).
Thus, there is a need for a peroxidase assay which is capable of detecting low levels of the enzyme, i.e., on the order of .ltoreq.10.sup.-7 M as are used in immunoassays. Such an assay desirably would be rapid and eliminate the requirement of the prior art fluorometric peroxidase assay system that the dichlorofluorescin be prepared as needed in a separate step. Furthermore, as with any similar assay technique, it is most desirable that all of the reagents can be stored in a stable, dry condition until immediately prior to use.